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Arkasubhra Ghosh, Vishnu Suresh Babu, Rayne Lim, Lisa Winer, Deepak SA, Ashwin Mallipatna, Shyam S Chaurasia, Nilanjan Guha; Retinoblastoma regulates cellular energetics through Hexokinase1 dependent AMPKα activation. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4962.
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© ARVO (1962-2015); The Authors (2016-present)
Biallelic inactivation of Retinoblastoma(Rb) tumor suppressor gene causes paediatric ocular tumors. Although the role of Rb in regulating cell cycle and tumor growth is well documented, the mechanisms regulating tumor cell energy metabolism need further investigation. This study investigates the Rb dependent molecular processes underlying energy sensing and associated regulatory mechanisms using an integrated multi-omics discovery platform and in vitro modelling.
Enucleated eyes of 9 patients & 2 pediatric deceased controls were used for multi-omics analysis. All the tissues were collected after written informed consent from the family and approved by Institutional Ethics Committee. WERI-Rb-1 cells (Weri) were used for 3D spheroid formation, proliferation, migration, chemosensitivity as well as for investigating molecular signalling by immunoblotting and quantitative PCR. Rb was complemented in Weri in the presence or absence of Hexokinase1 (HK1) and E2F2 using overexpression and shRNA approach. Agilent Seahorse XF was used for metabolic activity and stress assays.
Combined multi-omics analysis revealed ~1700 significantly modulated genes (FC ≥10, p ≤ 0.005). HK1 (FC=-17.46, p <0.005) and E2F2 (FC=540.2, p<0.005) were further analysed for their functional role in tumor formation. HK1 overexpression in Weri reduced 3D-spheroid formation, proliferation, migration, and chemosensitivity (p<0.05) compared to controls while E2F2 knockdown showed the similar results. The phenotypes of HK1 knockdown was partially complemented by Rb overexpression, similar to E2F2 overexpression. Rb complementation induced significant stabilization of HK1 protein and reduced E2F2 activity in immunoblots. Rb complemented Weri cells had reduced reserve respiratory capacity in Seahorse assays. Further, HK1 induced AMPKα phosphorylation without any change in its total protein levels. AMPKα phosphorylation correlated with reduction in ACC (acetyl-CoA carboxylase) levels. Rb overexpression in presence of HK1 knockdown did not activate AMPKα or reduce ACC. E2F2 modulation does not affect either AMPKα or ACC.
The data uncovers a unique signalling pathway wherein Rb protein reduces cellular spare respiratory capacity indicating a metabolic shift. We demonstrate that Rb dependent HK1 stabilisation activated AMPKα which in turn reduced ACC activation, a novel functional mechanism of Rb.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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